The proteins of the insulin family play essential roles in pleotropic physiological processes affecting metabolism, growth, and reproduction. Using the techniques of molecular and computational biology our laboratory recently identified a novel member of the insulin family we termed Insl6 (insulin-like protein 6). Expression of Insl6 is greatest in germ cells of the testis. Evolutionary conservation of the structure of the Insl6 gene suggests an essential biological role for this protein. However, at present the biological role of Insl6 is not known. Our preliminary studies have identified a subject with male infertility with a heterozygous mutation in the INSL6 gene. Our studies indicate that the identified mutation alters the intra-cellular trafficking of the Insl6 protein. Specific Aim 1 expands on these observations and tests the hypothesis that mutations in the INSL6 gene are associated with characteristic phenotypes in the human. The genotype-phenotype correlation will be established by analysis of clinical history, physical examination, sperm analysis, and endocrine profile. Specific Aim 2 tests the hypothesis that the Insl6 null phenotype, generated by targeted disruption of the murine Insl6 gene, will reflect the biological role of Insl6. The Insl6 null mice will be evaluated for phenotypic characteristics including viability, sexual differentiation, fertility, testicular macro- and microstructure, sperm analysis, and endocrine profile. The identification of new members of the insulin gene family has led to the elucidation of novel functions for this family of proteins. For example, targeted disruption of insulin-like peptide 3 (Insl3) in the mouse revealed a critical role for this peptide in testicular descent. Furthermore, the recent discovery of the cognate receptor (LGR8) for Insl3 and the elucidation of cryptorchidism as one of components of the LGR8 null phenotype underscore the exciting advances made in this field. The results from experiments outlined in this proposal will advance our understanding of the biological role of Insl6 and provide insights into human reproduction and disorders such as infertility.